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Volumn 284, Issue 5418, 1999, Pages 1359-1362

Stimulation of microtubule aster formation and spindle assembly by the small GTPase ran

Author keywords

[No Author keywords available]

Indexed keywords

GUANOSINE TRIPHOSPHATASE;

EID: 0033591373     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5418.1359     Document Type: Article
Times cited : (334)

References (29)
  • 8
    • 0029836330 scopus 로고    scopus 로고
    • R. Heald et al., Nature 382, 420 (1996).
    • (1996) Nature , vol.382 , pp. 420
    • Heald, R.1
  • 17
    • 0344487306 scopus 로고    scopus 로고
    • note
    • GST-Ran fusion proteins were purified and loaded with guanine nucleotide as described (16). Each protein was concentrated to 250 μM in ultrafree centrifugal filters (Millipore), then frozen in liquid nitrogen before storage at -80°C GST-Ran fusion proteins were diluted to 25 μM in CSF-arrested Xenopus egg extract for each assay. CSF-arrested Xenopus extracts and demembranated sperm were prepared as described (7, 14). The aster formation assay used was the same as described (24). Samples were viewed with a Nikon Eclipse E800 microscope and images captured with a cooled charge-coupled device (CCD) camera (Princeton Instruments) with the IP labs spectrum P software.
  • 22
    • 0344918449 scopus 로고    scopus 로고
    • note
    • To quantify the microtubule polymer mass in asters or spindles, we took images with a cooled CCD camera at the same exposure and below saturation level of the camera. Fluorescence intensity of each aster or spindle was measured, and the background was subtracted from the area next to each aster or spindle with the IP labs spectrum P software. Over 30 asters or spindles were measured in each experiment.
  • 26
    • 0344056101 scopus 로고    scopus 로고
    • note
    • Rabbit polyclonal antibodies were raised against a peptide corresponding to the last 15 amino acids of XMAP215 (D. Gard, personal communication) and affinity-purified against the peptide (23). These antibodies recognize XMAP215 by immunoblotting and specifically immunoprecipitate XMAP215 in Xenopus egg extracts (20). The extent of XMAP215 depletion was over 90%, as determined by protein immunoblotting.
  • 29
    • 0344056100 scopus 로고    scopus 로고
    • note
    • We thank L. Zhang and O. Martin for technical support; J. Gall, C. Wiese, and Q. Guo for critical reading the manuscript; members of the Zheng lab for helpful discussion and support; I. Macara (University of Virginia, Charlottesville) and K. Lounsbury (University of Vermont) for the Ran-GST fusion proteins; A. Merdes (University of Edinburgh) for the antibody to NuMA; and D. Card (University of Utah) for XMAP215 sequence information. Supported by NIH grant (GM55312-01) and a Pew Scholar's Award (Y.Z.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.